Morphological features and isoenzyme characterization of endosymbiotic algae from green hydra

Symbiotic associations are of a wide significance in evolution and biodiversity. Green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells, it harbors individuals of unicellular green algae. Morphological characteristics of isolated algae determined by light and electron microscopy are presented. Cytological morphometric parameters (cell area, cell radius, chloroplast area) of isolated algae from green hydra (Cx), as well as from reference species Chlorella kessleri (Ck) and Chlorella vulgaris (Cv), revealed similarity between the isolated endosymbiont and C. kessleri. Isoenzyme patterns of esterase (EST), peroxidase (POX), and catalase (CAT) were used for the investigation of genetic variability in endosymbiotic algae isolated from green hydra. Out of 14 EST isoenzymes observed in Cx species, 9 were expressed in the Cx sample. Results of the EST isoenzyme analysis indicated a higher degree of similarity between Cx and Cv than between Cx and Ck. Due to much higher heterogeneity, EST isoenzymes seem to be more suitable genetic markers for identification of different Chlorella species than CAT and POX isoenzymes. Results obtained suggest that symbiogenesis in green hydra has probably not been terminated yet.     

Interaction Between Equally Charged Membrane Surfaces Mediated by Positively and Negatively Charged Macro-Ions

In biological systems, charged membrane surfaces are surrounded by charged molecules such as electrolyte ions and proteins. Our recent experiments in the systems of giant phospholipid vesicles indicated that some of the blood plasma proteins (macro-ions) may promote adhesion between equally charged membrane surfaces. In this work, theory was put forward to describe an IgG antibody-mediated attractive interaction between negatively charged membrane surfaces which was observed in experiments on giant phospholipid vesicles with cardiolipin-containing membranes. The attractive interactions between negatively charged membrane surfaces in the presence of negatively and positively charged spherical macro-ions are explained using functional density theory and Monte Carlo simulations. Both, the rigorous solution of the variational problem within the functional density theory and the Monte Carlo simulations show that spatial and orientational ordering of macro-ions may give rise to an attractive interaction between negatively charged membrane surfaces. It is also shown that the distinctive spatial distribution of the charge within the macro-ions (proteins) is essential in this process.     

The Critical Roles of Residues P235 and F236 of Subunit A of the Motor Protein A-ATP Synthase in P-Loop Formation and Nucleotide Binding

The mutants P235A and F236A have been generated and their crystal structure was determined to resolutions of 2.38  and 2.35 Å, respectively, in order to understand the residues involved in the formation of the novel arched P-loop of subunit A of the A-ATP synthase from Pyrococcus horikoshii OT3. Both the structures show unique, altered conformations for the P-loop. Comparison with the previously solved wild type and P-loop mutant S238A structures of subunit A showed that the P-loop conformation for these two novel mutants occupy intermediate positions, with the wild type fully arched and the well-relaxed S238A mutant structures taking the extreme positions. Even though the deviation is similar for both mutants, the curvature of the P-loop faces the opposite direction. Deviations in the GER-loop, lying above the P-loop, are similar for both mutants, but in F236A, it moves towards the P-loop by around 2 Å. The curvature of the loop region V392-V410, located directly behind the P-loop, moves close by 3.6 Å towards the P-loop in the F236A structure and away by 2.5 Å in the P235A structure. Two major deviations were observed in the P235A mutant, which are not identified in any of the subunit A structures analyzed so far, one being a wide movement of the N-terminal loop region (R90-P110) making a rotation of 80° and the other being rigid-body rotation of the C-terminal helices from Q520-A588 by around 4° upwards. Taken together, the data presented demonstrate the concerted effects of the critical residues P235A, F236, and S238 in the unique P-loop conformation of the A-ATP synthases.     

Differential white blood cell count and type 2 diabetes: systematic review and meta-analysis of cross-sectional and prospective studies

Objective

Biological evidence suggests that inflammation might induce type 2 diabetes (T2D), and epidemiological studies have shown an association between higher white blood cell count (WBC) and T2D. However, the association has not been systematically investigated.

Research Design and Methods

Studies were identified through computer-based and manual searches. Previously unreported studies were sought through correspondence. 20 studies were identified (8,647 T2D cases and 85,040 non-cases). Estimates of the association of WBC with T2D were combined using random effects meta-analysis; sources of heterogeneity as well as presence of publication bias were explored.

Results

The combined relative risk (RR) comparing the top to bottom tertile of the WBC count was 1.61 (95% CI: 1.45; 1.79, p = 1.5*10⁻¹⁸). Substantial heterogeneity was present (I² = 83%). For granulocytes the RR was 1.38 (95% CI: 1.17; 1.64, p = 1.5*10⁻⁴), for lymphocytes 1.26 (95% CI: 1.02; 1.56, p = 0.029), and for monocytes 0.93 (95% CI: 0.68; 1.28, p = 0.67) comparing top to bottom tertile. In cross-sectional studies, RR was 1.74 (95% CI: 1.49; 2.02, p = 7.7*10⁻¹³), while in cohort studies it was 1.48 (95% CI: 1.22; 1.79, p = 7.7*10⁻⁵). We assessed the impact of confounding in EPIC-Norfolk study and found that the age and sex adjusted HR of 2.19 (95% CI: 1.74; 2.75) was attenuated to 1.82 (95% CI: 1.45; 2.29) after further accounting for smoking, T2D family history, physical activity, education, BMI and waist circumference.

Conclusions

A raised WBC is associated with higher risk of T2D. The presence of publication bias and failure to control for all potential confounders in all studies means the observed association is likely an overestimate.     

pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol

Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 degrees C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.     

Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: protein versus solvent environments

Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing “locked” chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these six systems is compared and spectral signatures of isomerization and solvation are discussed. We find that the ultrafast red-shifting of fluorescence is associated mostly with solvation dynamics, whereas isomerization manifests itself as quenching of fluorescence. The observed multiexponential quenching of the protein samples differs from the single-exponential lifetimes of the chromophores in solution. The locked chromophore in the protein environment decays faster than in solution. This is due to additional channels of excited-state energy dissipation via the covalent and hydrogen bonds with the protein environment. The observed large dispersion of quenching timescales observed in the protein samples that contain the native pigment favors both an inhomogeneous model and an excited-state barrier for isomerization.     

Effects of war aggression in Croatia on some forms and manifestations of breast cancer

The aim of this study was to determine the differences in epidemiological and clinical manifestations of breast cancer in time of war in Croatia and in peacetime before and after the war, in the defined population of Pozesko-Slavonska County. The methods used in this study were the evaluation of relative predictive value of variables by means of chi 2-test and the analysis of variance, while the survival studies were tested by Long Rank test according to Kaplan-Meier analysis of survival. This work encompasses 660 patients who had breast cancer. The differences between three time periods were evaluated: the war period (1991-1995), and two control periods: before the war (1981-1990) and after the war (1996-2000). The patients were grouped by age, localization of tumor and survival. During the war period the patients were, on the average, 4.2 years younger than those who acquired the disease in control periods, and the mean age of patients was between 50 and 59 years (36.5% of patients). Although the difference in tumor distribution by sides (left or right breast) and quadrants was statistically significant (war period vs. control periods; p < 0.001), there was no statistically significant difference in the survival of patients according to the results of Kaplan-Meier analysis of localization of tumor (p > 0.05). The length of survival in terms of age of patients in time of surgical procedure was significantly different (p < 0.001). This study confirms the effects of war on some epidemiological and clinical manifestation of breast cancer in the defined population of Pozesko-Slavonska County.     

C-H. . .pi interactions in the metal-porphyrin complexes with chelate ring as the H acceptor

Specific C-H. . .pi interactions with the pi-system of porphyrinato chelate ring were found in crystal structures of transition metal complexes from the Cambridge Structural Database and statistical analysis of geometrical parameters for intramolecular and intermolecular interactions was done. By density functional theory calculations on a model system it was evaluated that an interaction energy is above 1.5 kcal/mol and that the strongest interaction occurs when the distance between hydrogen atom and the center of the chelate ring is 2.6 A. This prediction is in good agreement with the distances for intermolecular interactions found in the crystal structures. In many cases the intramolecular interaction distances are much shorter than 2.6 A, and these short distances are caused by geometrical constrains. The C-H. . .pi interactions with chelate ring of porphyrinato ligand can influence the structure, contribute to its stability, and play some role in the function of biomolecules with metalo porphyrins.